Hyddroperoxide genes and tolerance to abiotic stress in plants

ABSTRACT

This invention provides for novel methods for preparing a plant tolerant to abiotic stress, such as drought or salt. This invention also provides for transgenic plants and transgenic seeds that are tolerant to abiotic stress. The methods of the present invention comprises introducing a recombinant expression cassette comprising a hydroperoxide lyase polynucleotide encoding a hydroperoxide lyase enzyme into the plants, and selecting a plant that is tolerant to abiotic stress. The transgenic plants and seeds generated by the methods of the invention accordingly comprise a recombinant expression cassette comprising a HPL polynucleotide encoding HPL enzyme.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant number0543904, awarded by the National Science Foundation. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The oxylipin pathway orchestrates a multitude of biological processes inresponse to developmental and environmental stimuli across the animaland plant kingdoms. The products of the oxylipin pathway are derivedfrom fatty acid oxidation and are designated as oxylipins. In plants,these compounds are mainly derived from the oxidation of α-linolenic(α-LeA: 18:3) and linoleic acids (LA: 18:2).

The biosynthesis of oxylipins is initiated by the action of lipases oncomplex membrane lipids causing the release of unesterified fatty acids.Subsequently, lipoxygenases (linoleate oxygen oxidoreductases, LOXs)introduce molecular oxygen to either the 9 or the 13 position of 18:2and 18:3, and convert them into their corresponding 9- or 13-hydroperoxyfatty acids [9/13-hydroperoxyoctadecatrienoic acid (9/13-HPOT) and9/13-hydroperoxyoctadecadienoic acid (9/13-HPOD)] (Dhondt, S. et al.,Plant J. 23:431-440, 2000; Vick, B. A., In: Moore, T. S., Lipidmetabolism in plants, CRC Press Inc., Florida, pp. 167-191, 1993; Brash,A. R., J. Biol. Chem. 274:23679-23682, 1999; Narvaez-Vasquez, J. et al.,Plant Cell 11:2249-2260, 1999). These hydroperoxides become thesubstrates for subsequent action of the four major metabolic pathwaysnamely, the peroxygenase (POX), divinyl ether synthase (DES), alleneoxide synthase (AOS) and hydroperoxide lyase (HPL) pathways (Feussner,I. and Wasternack, C., Annu. Rev. Plant Physiol. Plant Mol. Biol.53:275-297, 2002). Among these pathways, the AOS- and HPL-branches areconsidered to be the two major critical plant stress-response pathways.They compete for the same substrates and are responsible for theproduction of lipid-based signaling compounds, antimicrobial andantifungal compounds, and aromatic compounds (Feussner, I. andWasternack, C., supra; Howe, G. and Schilmiller, A. L., Curr. Opin.Plant Bio. 5:230-236, 2002). The AOS branch of 13-LOX transforms 13-HPOTto the jasmonate family of compounds that includes jasmonic acid (JA),methyl jasmonate (MeJA), and their metabolic precursor,12-oxo-phytodienoic acid (120PDA) (Howe, G. and Schilmiller, A. L.,supra).

Though Arabidopsis thaliana has one HPL, many plant species have morethan one gene encoding HPL enzymes. For example, Medicago truncatula isreported to have two, and alfalfa and rice each have three HPLs(Noordermeer, M. A. et al., Eur. J. Biochem. 267:2473-2482, 2000;Chehab, E. W. et al., Plant Physiol. 141:121-134, 2006). This variationin the number of genes among plant species may reflect the differentialregulation of this pathway and, ultimately, the diversity of thespecies' responses to various stimuli.

HPL enzymes catalyze the cleavage of 9/13-hydroperoxides and produce arange of metabolites. The action of HPL on 9-HPOT/HPOD gives rise to thebactericidal C9 aldehydes and oxoacids involved in the flavors and odorsof fruits and leaves (Vick, B. A., In: Moore, T. S., Lipid metabolism inplants, CRC Press Inc., Florida, pp. 167-191, 1993; Brash, A. R., J.Biol. Chem. 274:23679-23682, 1999; Matsui, K., Curr. Opin. Plant Biol.9(3):274-280, 2006; Cho, M. J. et al., J. Food Prot. 67:1014-1016,2004). Activity of HPL on 13-HPOT/HPOD leads to the production of thegreen leaf volatiles (GLVs) that are comprised of Z-3-hexenal andn-hexanal, and their corresponding alcohols, generated through theaction of alcohol dehydrogenase (ADH), and esters, respectively (Matsui,K., Curr. Opin. Plant Biol. 9(3):274-280, 2006). An acyl-transferase(CHAT) converts Z-3-hexenol to Z-3-hexenyl acetate (d'Auria, J. C. etal., Plant J. 49:194-207, 2006). In addition, isomerization ofZ-3-hexenal results in generation of E-2-hexenal.

It has been shown that the presence of three rice HPL genes (HPL1, HPL2,and HPL3) are distinct in their levels and patterns of expression(Chehab, E. W. et al., Plant Physiol. 141(1):121-34, 2006). The threecorresponding encoded enzymes also differ in their substrate specificityas determined by in vitro enzyme assays, in conjunction with therespective profiles of their cognate metabolites in transgenicArabidopsis generated in the Columbia-0 ecotype (Col-0) background. TheCol-0 ecotype is a natural hpl mutant that expresses the gene transcriptbut because of a 10 base pair deletion encodes a dysfunctional enzymeand thus lacks C6-aldehydes (Duan, H. et al., Plant Physiol.139:1529-1544, 2005).

The role of aldehydes generated by overexpression of rice HPL3 invarious backgrounds has been examined (Chehab, E. W. et al., PLoS ONE3(4): e1904, 2008). It has been shown that hexenyl acetate is thepredominant wound-inducible volatile signal that mediates indirectdefense responses by directing tritrophic (plant-herbivore-naturalenemy) interactions.

However, the role of these metabolic pathways, and of the hydroperoxidelyases in particular, on plant stress-responses besides responses towounding and insect damage is not well understood in the prior art. Thisand other problems are addressed by the present invention.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to the development of abioticstress-tolerant plants. In accordance with various embodiments of theinvention, methods of preparing plants with increased abioticstress-tolerance and/or other advantageous characteristics—such as, forexample, increased biomass, increased seed yield, heavier grains, alonger grain-filling period, and/or sturdier stems—are provided. Inaccordance with an exemplary embodiment, this invention is directed tothe preparation of transgenic plants that express a hydroperoxide lyasesequence, and preferably, a heterologous hydroperoxide lyase sequence.

The methods of the invention comprise introducing into a population ofplants a recombinant expression cassette comprising a hydroperoxidelyase (HPL) polynucleotide encoding a HPL enzyme; and selecting a plantthat is tolerant to abiotic stress, wherein the HPL enzyme comprises(L/I)-(F/C)-G-(Y/F)-(Q/R)-(P/K), wherein the HPL enzyme furthercomprises (N/D)-K-(Q/I)-C-(A/P)-(G/A)-K-(D/N). The step of introducingthe expression cassette can be carried out using any known method. Forexample, the expression cassette can be introduced byAgrobacterium-mediated transformation of plant cells, a sexual cross orusing micro-projectile bombardment of plant cells.

In accordance with one aspect of an exemplary embodiment of theinvention, the HPL enzyme is localized outside the plastid whenexpressed in the population of plants. In some embodiments of theinvention, the HPL enzyme recognizes 9-hydroperoxy-octadecatrienoic acid(9-HPOT) or 9-hydroperoxy-octadecadienoic acid (9-HPOD). In someembodiments of the invention, the HPL enzyme recognizes13-hydroperoxy-octadecatrienoic acid (13-HPOT) or13-hydroperoxy-octadecadienoic acid (13-HPOD).

In some embodiments of the invention, the HPL enzyme is localizedoutside the plastid when expressed in the population of plants, andwherein the HPL enzyme recognizes 9-hydroperoxy-octadecatrienoic acid(9-HPOT) or 9-hydroperoxy-octadecadienoic acid (9-HPOD). In someembodiments of the invention, the HPL enzyme further recognizes13-hydroperoxy-octadecatrienoic acid (13-HPOT) or13-hydroperoxy-octadecadienoic acid (13-HPOD).

In accordance with one exemplary embodiment of the invention, a methodof preparing a plant tolerant to abiotic stress comprises introducinginto a population of plants a recombinant expression cassette comprisinga hydroperoxide lyase (HPL) polynucleotide encoding a HPL enzyme whereinthe HPL enzyme has an amino acid sequence at least 90% identical to SEQID NO. 2, 4 or 6, and selecting a plant that is tolerant to abioticstress, wherein the HPL enzyme is localized extraplastidially whenexpressed in the population of plants, and wherein the HPL enzymerecognizes 9-hydroperoxy-octadecatrienoic acid (9-HPOT) or9-hydroperoxy-octadecadienoic acid (9-HPOD), and further recognizes13-hydroperoxy-octadecatrienoic acid (13-HPOT) or13-hydroperoxy-octadecadienoic acid (13-HPOD).

In some embodiments of the invention, the abiotic stress is drought. Insome embodiments of the invention, the abiotic stress is salinity.

In some embodiments of the invention, the HPL polynucleotide is operablylinked to a promoter. The promoter of choice could be either aconstitutive promoter, or an inducible promoter, or a tissue-preferredpromoter.

In some embodiments of the invention, the HPL enzyme has an amino acidsequence at least 90% identical to SEQ ID NO. 2, 4 or 6. In someembodiments of the invention, the HPL enzyme has an amino acid sequenceat least 91%, 92%, 93%, 94%, or 95% identical to SEQ ID NO. 2, 4 or 6.In some embodiments of the invention, the HPL enzyme has an amino acidsequence at least 96%, 97%, 98%, or 99% identical to SEQ ID NO. 2, 4 or6. In some embodiments of the invention, the amino acid sequenceidentity of the HPL enzyme to SEQ ID NO. 2, 4 or 6 may be lower than 90%provided that the HPL enzyme comprises (L/I)-(F/C)-G-(Y/F)-(Q/R)-(P/K)and (N/D)-K-(Q/I)-C-(A/P)-(G/A)-K-(D/N). In some embodiments of theinvention, the HPL polynucleotide is SEQ ID NO. 1, 3 or 5.

The HPL polynucleotide can be introduced into any plant capable oftransformation with recombinant expression constructs. The expression inOryza sativa is preferred herein. In accordance with various exemplaryembodiments of this invention, other dicots or monocots may be utilizedwith comparable utility.

The present invention also relates to abiotic stress-tolerant transgenicplants. The transgenic plants of the invention have increased abioticstress-tolerance and/or other advantageous characteristics, such as, forexample, increased biomass, increased seed yield, heavier grains, alonger grain-filling period, and/or sturdier stems. This invention isdirected to transgenic plants that express a hydroperoxide lyase.

The transgenic plants of the invention comprise a recombinant expressioncassette comprising a HPL polynucleotide encoding a HPL enzyme or anyactive fragment thereof having an amino acid sequence either identicalto SEQ ID NO. 2, 4 or 6 or with sufficient identity to SEQ ID NO. 2, 4or 6 to achieve similar functionality as SEQ ID NO. 2, 4 or 6, whereinthe transgenic plants are not Arabidopsis. An example of such transgenicplants is Oryza sativa.

The present invention also relates to transgenic seeds from thetransgenic plants of the invention. An example of such transgenic seedsis a transgenic Oryza sativa seed from the transgenic plants of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Survival test of HPL1 and HPL2 lines exposed to 200 mM salt.Data are shown as the mean±the standard deviation.

FIG. 2: Survival test of HPL lines exposed to drought. The linesoverexpressing HPL1 through 3 are designated as OsHPL1 OE, OsHPL2 OE andOsHPL3 OE, respectively; the line overexpressing HP-3 minus the first 15amino acids of the plastid transit peptide at the amino terminus of theenzyme is designated as OsHPL3-TP OE. Data are shown as the mean±thestandard deviation.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “HPL polynucleotide” refers to a polynucleotide that is derivedfrom the gene encoding the hydroperoxide lyase polypeptide and encodes apolypeptide that retains hydroperoxide lyase enzymatic activity. HPLencodes hydroperoxide lyase. Several HPL genes have been isolated fromrice including, HPL1, HPL2, and HPL3. The term as used hereinencompasses a polynucleotide including a native hydroperoxide lyasesequence, as well as modifications and fragments thereof. The term HPLpolynucleotide as used herein encompass a polynucleotide including,respectively, a native hydroperoxide lyase sequence as well asmodifications and fragments that code for an active HPL polypeptide.

The term “HPOT” refers to hydroperoxy-octadecatrienoic acid. The term“HPOD” refers to hydroperoxy-octadecadienoic acid. The term “9-HPOT”refers to 9-hydroperoxy-octadecatrienoic acid. The term “9-HPOD” refersto 9-hydroperoxy-octadecadienoic acid. The term “13-HPOT” refers to13-hydroperoxy-octadecatrienoic acid. The term “13-HPOD” refers to13-hydroperoxy-octadecadienoic acid.

The term “polypeptide” refers to a polymer of amino acids and caninclude full-length proteins, polypeptide, and fragments thereof. In thepresent invention, “HPL polypeptide” means a polypeptide having at leastone HPL function.

Thus, the term “HPL polynucleotide” and “HPL polypeptide” of theinvention may include alterations to the polynucleotide or polypeptidesequences, so long as the alteration results in a molecule displayingHPL activity. Thus, the polynucleotide or polypeptide may besubstantially identical to a reference sequence (e.g., SEQ ID NOs: 1-6).The sequence identity may be lower than 90% provided that the HPL enzymecomprises (L/I)-(F/C)-G-(Y/F)-(Q/R)-(P/K) and(N/D)-K-(Q/I)-C-(A/P)-(G/A)-K-(D/N). Whereas some native HPL moleculesare localized in the plastid, some are localized outside the plastid. Agood way to localize a polypeptide that would normally be localizedinside the plastid extraplastidially is to remove the first 15 aminoacids of its plastid transit peptide or to fuse it at the amino terminalto another protein and confirm that it is not localized to the plastid.Removal of the transit peptide should not affect enzyme activity;however, the activity displayed by some mutant molecules may not at thesame level as the native molecule. Modifications of the polynucleotidesequences described herein typically include deletions, additions andsubstitutions, to the native HPL sequences. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of plants that express the polynucleotide orerrors due to PCR amplification. The term encompasses expressed allelicvariants of the wild-type sequence which may occur by normal geneticvariation or are produced by genetic engineering methods and whichresult in HPL activity.

The term “heterologous” in reference to a nucleic acid or polynucleotideof the present invention refers to a nucleic acid or polynucleotide thatoriginates from a foreign species, or if from the same species, isaltered in some way (e.g., mutated, added in multiple copies, linked toa non-native promoter or enhancer sequence, etc.).

The term “plastids” mean the organelles in plants including but notlimited to chloroplasts and chromoplasts.

The term “progeny” refers generally to the offspring of a cross andincludes direct F1 progeny, as well as later generations of F2, F3, etc.

The term “introgression” refers generally to the movement of a gene fromone species into the gene pool of another by genetic crosses. Generally,this is accomplished by repeated backcrossing of an interspecific hybridwith one of its parents.

As used herein, the term “abiotic stress” or “abiotic stress condition”refers to the exposure of a plant, plant cell, or the like, to anon-living (“abiotic”) physical or chemical agent or condition that hasan adverse effect on metabolism, growth, development, propagation and/orsurvival of the plant (collectively “growth”). Abiotic stress can beimposed on a plant due, for example, to an environmental factor such asexcessive or insufficient water (e.g., flooding, drought, dehydration),anaerobic conditions (e.g., a low level of oxygen), abnormal osmoticconditions, salinity or temperature (e.g., hot/heat, cold, freezing,frost), a deficiency of nutrients or exposure to pollutants, or by ahormone, second messenger or other molecule. Anaerobic stress, forexample, is due to a reduction in oxygen levels (hypoxia or anoxia)sufficient to produce a stress response. A flooding stress can be due toprolonged or transient immersion of a plant, plant part, tissue orisolated cell in a liquid medium such as occurs during monsoon, wetseason, flash flooding or excessive irrigation of plants, or the like. Acold stress or heat stress can occur due to a decrease or increase,respectively, in the temperature from the optimum range of growthtemperatures for a particular plant species. Such optimum growthtemperature ranges are readily determined or known to those skilled inthe art. Dehydration stress can be induced by the loss of water, reducedturgor, or reduced water content of a cell, tissue, organ or wholeplant. Drought stress can be induced by or associated with thedeprivation of water or reduced supply of water to a cell, tissue, organor organism. Saline stress (salt-stress) can be associated with orinduced by a perturbation in the osmotic potential of the intracellularor extracellular environment of a cell. Osmotic stress also can beassociated with or induced by a change, for example, in theconcentration of molecules in the intracellular or extracellularenvironment of a plant cell, particularly where the molecules cannot bepartitioned across the plant cell membrane.

A plant's response to abiotic stress includes the production of excessreactive oxygen species (ROS), including singlet oxygen, superoxide,hydrogen peroxide and hydroxyls radicals, which act as signalingmolecules and play a role in the initiation of defense mechanisms. ROSare involved in diverse environmental stress in plants. Excessivetemperature extremes, water stress, ion imbalances due to salinity, airpollution, and mechanical damage (such as wounding by sucking or chewinginsects or breakage due to wind, etc.) lead to chemical signalspropagated through ROS. Adaptation to the stress will involve aquenching of ROS signal through on or more anti-oxidant enzymes orcompounds, such as superoxide dismutase (SOD), glutathione, ascorbate,carotenoids, and others. When the plants quenching systems are exceededby the environmental stress, extensive and rapid degeneration reactionscan occur through ROS, such as protein denaturation and lipidperoxidation. The improved tolerance to one particular type of abioticstress, such as drought, may confer a similarly improved tolerance toanother, such as high light or heat, when part of the mechanism ofimproved tolerance includes improved quenching of oxidative or ROSstress.

Plants suffer heat stress when temperatures are hot enough for a longenough period of time to cause irreversible damage to plant function,development and/or yield. Heat stress can have detrimental effects onreproductive development and reduce yield (abnormal biomass and/or fruitand seed). When subjected to extreme heat stress, plants may notsurvive.

The invention provides a genetically modified plant, which can be atransgenic plant, that is more tolerant to a stress condition than acorresponding reference plant. As used herein, the term “tolerant” whenused in reference to a stress condition of a plant, means that theparticular plant, when exposed to a stress condition, shows less of aneffect, or no effect, in response to the condition as compared to acorresponding reference plant (naturally occurring wild-type plant or aplant not containing a construct of the present invention). As aconsequence, a plant encompassed within the present invention showsimproved agronomic performance as a result of enhanced abiotic stresstolerance and grows better under more widely varying conditions, such asincreased biomass and/or higher yields and/or produces more seeds.Preferably, the transgenic plant is capable of substantially normalgrowth under environmental conditions where the corresponding referenceplant shows reduced growth, yield, metabolism or viability, or increasedmale or female sterility.

As used herein, the term “drought-tolerance” refers to the moredesirable productivity of a plant under conditions of water deficitstress. Water deficit stress develops as the evapotranspiration demandfor water exceeds the supply of water. Water deficit stress can be oflarge or small magnitude (e.g., days or weeks of little or no accessiblewater), but drought tolerant plants will show better growth and/orrecovery from the stress, as compared to drought sensitive plants.

As used herein, the term “water use efficiency” refers to the moredesirable productivity of a plant per unit of water applied. The appliedwater may be the result of precipitation or irrigation.

As used herein, the term “salt-tolerance” refers to the more desirableproductivity of a plant under conditions of salinity stress. While foreach species, the threshold at which soil and/or water salinity (oftenexpressed as conductivity, or E.C.) differs, a salt-tolerant plant wouldhave a higher salinity threshold before yields decline. Salt-tolerancealso refers to the sensitivity of yield to water and/or soil salinitybeyond the threshold. So a salt-tolerant plant would show less impact onyield per unit of salinity (E.C.) than a salt-sensitive plant.Salt-tolerance refers to an increased threshold and/or a decreasedsensitivity beyond the threshold of yield to salinity.

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g., leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g., bracts, sepals, petals, stamens,carpels, anthers and ovules), seed (including embryo, endosperm, andseed coat) and fruit (the mature ovary), plant tissue (e.g., vasculartissue, ground tissue, and the like) and cells (e.g., guard cells, eggcells, trichomes and the like), and progeny of same. The class of plantsthat can be used in the method of the invention is generally as broad asthe class of higher and lower plants amenable to transformationtechniques, including angiosperms (monocotyledonous and dicotyledonousplants), gymnosperms, ferns, and multicellular algae. It includes plantsof a variety of ploidy levels, including aneuploid, polyploid, diploid,haploid and hemizygous.

As used herein, “transgenic plant” includes reference to a plant thatcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid, including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

The term “expression cassette” refers to any recombinant expressionsystem for the purpose of expressing a nucleic acid sequence of theinvention in vitro or in vivo, constitutively or inducibly, in any cell,including, in addition to plant cells, prokaryotic, yeast, fungal,insect or mammalian cells. The term includes linear and circularexpression systems. The term includes all vectors. The cassettes canremain episomal or integrate into the host cell genome. The expressioncassettes can have the ability to self-replicate or not (i.e., driveonly transient expression in a cell). The term includes recombinantexpression cassettes that contain only the minimum elements needed fortranscription of the recombinant nucleic acid.

The term “constitutive” or “constitutively” denotes temporal and spatialexpression of the polypeptides of the present invention in plants in themethods according to various exemplary embodiments of the invention. Theterm “constitutive” or “constitutively” means the expression of thepolypeptides of the present invention in the tissues of the plantthroughout the life of the plant and in particular during its entirevegetative cycle. In some embodiments, the polypeptides of the presentinvention are expressed constitutively in all plant tissues. In someembodiments, the polypeptides of the present invention are expressedconstitutively in the roots, the leaves, the stems, the flowers, and/orthe fruits. In other embodiments of the invention, the polypeptides ofthe present invention are expressed constitutively in the roots, theleaves, and/or the stems.

The term “inducible” or “inducibly” means the polypeptides of thepresent invention are not expressed, or are expressed at very lowlevels, in the absence of an inducing agent. The expression of thepolypeptides of the present invention is greatly induced in response toan inducing agent.

The term “inducing agent” is used to refer to a chemical, biological orphysical agent or environmental condition that effects transcriptionfrom an inducible regulatory element. In response to exposure to aninducing agent, transcription from the inducible regulatory elementgenerally is initiated de novo or is increased above a basal orconstitutive level of expression. Such induction can be identified usingthe methods disclosed herein, including detecting an increased level ofRNA transcribed from a nucleotide sequence operatively linked to theregulatory element, increased expression of a polypeptide encoded by thenucleotide sequence, or a phenotype conferred by expression of theencoded polypeptide.

The term “homolog” is used to refer to a gene that is similar instructure and evolutionary origin to a gene in another species. In thecase of HPL genes, homologs encode proteins that belong to thecytochrome P450 family and contain the typical signature domainsdesignated as I-, K-helices and the Heme-binding domain, and thatcatalyze the cleavage of 9/13-hydroperoxides to produce thecorresponding metabolites, including, but not limited to, C9 aldehydesand oxoacids from 9-hydroperoxy-octadecatrienoicacids/hydroperoxy-octadecadienoic acids and C8 aldehydes, hexenals andhexanals, from 13-hydroperoxy-octadecatrienoicacids/hydroperoxy-octadecadienoic acids. See Chehab et al. (J.Integrative Plant Biol. 49(1):43-51, 2007) for a description of thephylogenetic analysis and sequence alignments of HPL homologs fromseveral species showing the HPL consensus sequences(L/I)-(F/C)-G-(Y/F)-(Q/R)-(P/K) and (N/D)-K-(Q/I)-C-(A/P)-(G/A)-K-(D/N).As genomic sequences become available, methods known in the art can beused to identify additional HPL homologs from other species.

The phrase “substantially identical,” in the context of the presentinvention refers to polynucleotides or polypeptides that have sufficientsequence identity with a reference sequence (e.g., one of SEQ ID NOs:1-6) to effect similar functionality when expressed in plants as thereference sequence. In accordance with one aspect of an exemplaryembodiment of the invention, a polynucleotide or a polypeptide thatexhibits at least 90% sequence identity with a reference sequence (e.g.,one of SEQ ID NOs: 1-6) may be deemed to be “substantially identical;”however, polynucleotides and polypeptides that exhibit less (evensignificantly less, e.g., 60%-70% or less) than 90% sequence identitymay, in accordance with various exemplary embodiments of the invention,be “substantially identical” to their reference sequences if requisitefunctionality is achieved. Alternatively, percent identity can be anyvalue from 90% to 100%. More preferred embodiments include at least:90%, 95%, or 99% identity as used herein is as compared to the referencesequence using the programs described herein; preferably BLAST usingstandard parameters, as described below. The sequence identity of thepolynucleotides and plypeptides may be lower than 90% provided that theHPL enzyme comprises (L/I)-(F/C)-G-(Y/F)-(Q/R)-(P/K) and(N/D)-K-(Q/I)-C-(A/P)-(G/A)-K-(D/N).

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions, such as from 20 to600, usually about 50 to about 200, more usually about 100 to about 150,in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. If no range is provided, the comparison window is the entirelength of the reference sequence. Methods of alignment of sequences forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted [e.g., by the local homology algorithm ofSmith and Waterman, Adv. Appl. Math. 2:482, 1981; by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970;by the search for similarity method of Pearson and Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988; by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection].

An example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul, S. F. et al., J. Mol. Biol. 215:403-410, 1990.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul, S. F. et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a wordlength (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl.Acad. Sci. USA 89:10915, 1989), alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, preferably less thanabout 0.01, and more preferably less than about 0.001.

II. Nucleic Acids

In accordance with one aspect of an exemplary embodiment of the presentinvention, a polynucleotide may include (a) a polynucleotide encoding apolypeptide of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, includingexemplary polynucleotides of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO:5; (b) a polynucleotide having a specified sequence identity withpolynucleotides of (a); (c) homologs of SEQ ID NOs: 1, 3 and 5; (d)complementary sequences of polynucleotides of (a), (b) or (c); and (e)active fragments of any of (a), (b), (c) or (d).

The present invention provides, among other things, isolated nucleicacids of RNA, DNA, and analogs and/or chimeras thereof, comprising apolynucleotide of the present invention.

A. Polynucleotides Encoding a Polypeptide of the Present Invention

The present invention provides isolated nucleic acids comprising apolynucleotide of the present invention, wherein the polynucleotideencodes a polypeptide of the present invention or an active fragmentthereof. Every nucleic acid sequence herein that encodes a polypeptidealso, by reference to the genetic code, describes every possible silentvariation of the nucleic acid. One of ordinary skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine; and UGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Thus, each silent variation of a nucleic acid, which encodes apolypeptide of the present invention, is implicit in each describedpolypeptide sequence and is within the scope of the present invention.Accordingly, the present invention includes polynucleotides of thepresent invention and polynucleotides encoding a polypeptide of thepresent invention.

B. Polynucleotides Having a Specific Sequence Identity with thePolynucleotides of (A)

In accordance with various exemplary embodiments, the present inventionprovides isolated HPL nucleic acids comprising HPL polynucleotides asdiscussed herein above, wherein the HPL polynucleotides have a specifiedidentity at the nucleotide level to a polynucleotide as disclosed abovein section (A) above. Percent identity can be calculated using, forexample, the BLAST algorithm under default conditions.

C. Polynucleotides That Are Homologs

The present invention provides isolated HPL nucleic acids comprising HPLnucleotides that are homologs of SEQ ID NOs: 1, 3 and 5. Some HPLhomologs are described in Chehab et al. (J. Integrative Plant Biol.49(1):43-51, 2007), others can be identified using methods known in theart, and as additional genomic sequences become available additional HPLhomologs from other species can be identified.

D. Polynucleotides Complementary to the Polynucleotides of (A)-(C)

The present invention provides isolated nucleic acids comprisingpolynucleotides complementary to the polynucleotides of sections A-B,above. As those of skill in the art will recognize, complementarysequences base pair throughout the entirety of their length with thepolynucleotides of sections (A)-(C) (i.e., sequences that are 100%complementary over their entire length). Complementary bases associatethrough hydrogen bonding in double stranded nucleic acids. For example,the following base pairs are complementary: guanine and cytosine;adenine and thymine; and adenine and uracil. Moreover, those skilled inthe art will recognize that sequences that base pair throughout theentirety of their regions of overlap (i.e., are 100% complementary inoverlapping regions, but are not 100% complementary over their entirelength) may be complementary. Furthermore, sequences that are not 100%complementary can still work as anti-sense constructs, and thus mayachieve the stated function of this aspect of the inventionnotwithstanding lesser complementarity (e.g., 60%-70% or less).

III. Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made usingstandard recombinant methods, synthetic techniques, combinationsthereof, or any other method now known or hereafter developed forpreparing such nucleic acids.

A. Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention can be obtainedfrom plant biological sources (e.g., tissues from the plant) using anynumber of cloning methodologies now known to or hereafter devised bythose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize under stringent conditions to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. Isolation of RNA andconstruction of cDNA and genomic libraries is well known to those ofordinary skill in the art. See, e.g., Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin, 1997; andCurrent Protocols in Molecular Biology, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York, 1995.

A1. Genomic DNA

The isolated nucleic acid compositions of this invention can be obtaineddirectly from genomic DNA isolated from Oryza sativa (Chehab, et al.,Plant Phys. 141: 121-134, 2006).

A2. cDNA Libraries

A number of cDNA synthesis protocols have been described that provideenriched full-length cDNA libraries. Enriched full-length cDNA librariesare constructed to comprise at least 60%, and more preferably at least70%, 80%, 90% or 95% full-length inserts amongst clones containinginserts. The length of insert in such libraries can be at least 2, 3, 4,5, 6, 7, 8, 9, 10 or more kilobase (kb) pairs. Vectors to accommodateinserts of these sizes are known in the art and available commercially.See, e.g., Stratagene's lambda ZAP Express (cDNA cloning vector with 0to 12 kb cloning capacity). An exemplary method of constructing agreater than 95% pure full-length cDNA library is described by Carninciet al., Genomics 37:327-336, 1996. Other methods for producingfull-length libraries are known in the art. See, e.g., Edery et al.,Mol. Cell Biol. 15(6):3363-3371, 1995; and PCT ApplicationWO/1996/034981.

A non-normalized or subtracted cDNA library also can be used forconstructing nucleic acids of the present invention according tostandard protocols.

The cDNA or genomic library can be screened using a probe based upon thesequence of a HPL polynucleotide of the present invention such as thosedisclosed herein. Probes may be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentplant species. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent.

The nucleic acids of interest can also be amplified from nucleic acidsamples using amplification techniques. For instance, polymerase chainreaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing, or for other purposes. The T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

PCR-based screening methods have been described. Wilfinger et al.describe a PCR-based method in which the longest cDNA is identified inthe first step so that incomplete clones can be eliminated from study(Bio Techniques 22(3): 481-486, 1997). Such methods are particularlyeffective in combination with a full-length cDNA constructionmethodology, above.

B. Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention also can be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang et al., Meth. Enzymol. 68: 90-99, 1979; thephosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151, 1979;the diethylphosphoramidite method of Beaucage et al., Tetra. Letts. 22:1859-1862, 1981; the solid phase phosphoramidite triester methoddescribed by Beaucage and Caruthers, Tetra. Letts. 22(20): 1859-1862,1981; e.g., using an automated synthesizer, e.g., as described inNeedham-VanDevanter et al., Nucleic Acids Res. 12: 6159-6168, 1984; and,the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesisgenerally produces a single stranded oligonucleotide. This may beconverted into double stranded DNA by hybridization with a complementarysequence, or by polymerization with a DNA polymerase using the singlestrand as a template. One of skill will recognize that while chemicalsynthesis of DNA is best employed for sequences of about 100 bases orless, longer sequences may be obtained by the ligation of shortersequences.

IV. Recombinant Expression Cassettes

In accordance with another aspect of an exemplary embodiment, thepresent invention provides recombinant expression cassettes comprising anucleic acid of the present invention. A nucleic acid sequence codingfor the desired polynucleotide of the present invention, for example acDNA or a genomic sequence encoding a full length polypeptide of thepresent invention, can be used to construct a recombinant expressioncassette, which can be introduced into the desired host cell. Arecombinant expression cassette will typically comprise a polynucleotideof the present invention operably linked to transcriptional initiationregulatory sequences, which will direct the transcription of thepolynucleotide in the intended host cell, such as tissues of atransformed plant.

For example, plant expression vectors may include (1) a cloned plantgene under the transcriptional control of 5′ and 3′ regulatory sequencesand (2) a dominant selectable marker. Such plant expression vectors mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

A. Vectors

Typical vectors useful for expression of genes in higher plants are wellknown in the art. A number of expression vectors suitable for stabletransformation of plant cells or for the establishment of transgenicplants have been described including those described in Weissbach andWeissbach, Methods for Plant Molecular Biology, Academic Press, 1989,and Gelvin et al., Plant Molecular Biology Manual, Kluwer AcademicPublishers, 1990. Specific examples include those derived from atumor-inducing (Ti) plasmid or a root-inducing (Ri) plasmid ofAgrobacterium tumefaciens, as well as those disclosed byHerrera-Estrella, L., et al. (Nature 303:209, 1983), Bevan, M. (Nucl.Acids Res. 12: 8711-8721, 1984) and Klee, H. J. (Bio/Technology3:637-642, 1985) for dicotyledonous plants. Ti-derived plasmids can betransferred into both monocotonous and docotyledonous species usingAgrobacterium-mediated transformation (Ishida et al., Nat. Biotechnol.14:745-50, 1996; Barton et al., Cell 32:1033-1043, 1983). ExemplaryAgrobacterium tumefaciens vectors useful herein are plasmids pKYLX6 andpKYLX7 of Schardl, et al. (Gene 61:1-11, 1987) and Berger et al. (Proc.Natl. Acad. Sci. USA 86:8402-6, 1989). Another useful vector herein isplasmid pBI101.2 that is available from CLONTECH Laboratories, Inc.(Palo Alto, Calif.).

Alternatively, non-Ti vectors can be used to transfer the DNA intoplants and cells by using free DNA delivery techniques. Such methods mayinvolve, for example, the use of liposomes, electroporation,microprojectile bombardment, silicon carbide whiskers, and viruses. Animmature embryo can also be a good target tissue for direct DNA deliverytechniques by using the particle gun (Weeks, T. et al., Plant Physiol.102:1077-1084, 1993; Vasil, V., Bio/Technology 10:667-674, 1993; Wan, Y.and Lemeaux, P., Plant Physiol. 104:37-48, 1994) and forAgrobacterium-mediated DNA transfer (Ishida et al., Nature Biotech.14:745-750, 1996).

B. Promoters B1. Constitutive Promoters

A number of promoters can be used in the practice of the invention. Aplant promoter fragment can be employed which will direct expression ofa polynucleotide of the present invention in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and state of development or cell differentiation. Examples ofconstitutive promoters include the cauliflower mosaic virus (CaMV) 35Stranscription initiation region.

B2. Inducible Promoters

Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention under environmental control.Such promoters are referred to here as “inducible” promoters.Environmental conditions that may effect transcription by induciblepromoters include biotic stress, abiotic stress, saline stress, droughtstress, pathogen attack, anaerobic conditions, cold stress, heat stress,hypoxia stress or the presence of light.

Examples of inducible promoters include, but are not limited to, asalt-inducible promoter rd29A (Kasuga, M. et al., Nature Biotechnol. 17,287-291, 1999), the drought-inducible promoter of maize (Busk et al.,Plant J. 11:1285-1295, 1997); the cold, drought, and high salt induciblepromoter from potato (Kirch, Plant Mol. Biol. 33:897-909, 1997), alight-inducible promoter PPDK, a light-inducible promoter from the smallsubunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), a hypoxiaor cold stress-inducible promoter Adh1, a heat stress-inducible promoterHsp70 promoter, and many cold inducible promoters known in the art, forexample rd29a and cor15a promoters from Arabidopsis thaliana (GenBankID: D13044 and U01377), blt1O1 and blt4.8 from barley (GenBank ID:AJ310994 and U63993), wcs120 from wheat (GenBank ID: AF031235), andmlipl5 from corn (GenBank ID: D26563).

Other inducible promoters that have been described include the ABA- andturgor-inducible promoters, the promoter of the auxin-binding proteingene (Schwob et al., Plant J. 4(3):423-432, 1993), the UDP glucoseflavonoid glycosyl-transferase gene promoter (Ralston et al., Genetics119:185-197, 1988), the MPI proteinase inhibitor promoter (Cordero etal., Plant J. 6(2):141-150, 1994), and the glyceraldehyde-3-phosphatedehydrogenase gene promoter (Kohler et al., Plant Mol. Biol.29(6):1293-1298, 1995; Quigley et al., J. Mol. Evol. 29(5):412-421,1989; Martinez et al., J. Mol. Biol. 208(4):551-565, 1989).

B3. Tissue-Preferred Promoters

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues, suchas leaves, roots, fruit, seeds, or flowers. These promoters aresometimes called tissue-preferred promoters. Exemplary promoters includethe anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and5,689,051), glob-1 promoter, and gamma-zein promoter. An exemplarypromoter for leaf- and stalk-preferred expression is MS8-15 (PCTPublication No. WO 98/00533). Examples of seed-preferred promotersincluded, but are not limited to, 27 kD gamma zein promoter and waxypromoter (Boronat, A. et al., Plant Sci. 47:95-102, 1986; Reina, M. etal, Nucleic Acids Res. 18(21):6426, 1990; and Kloesgen, R. B. et al.,Mol. Gen. Genet. 203:237-244, 1986). Promoters that express in theembryo, pericarp, and endosperm are disclosed in PCT Publication Nos. WO00/11177 and WO 00/12733 both of which are hereby incorporated byreference. The operation of a promoter may also vary depending on itslocation in the genome. Thus, a developmentally regulated promoter maybecome fully or partially constitutive in certain locations. Adevelopmentally regulated promoter can also be modified, if necessary,for weak expression.

Both heterologous and non-heterologous (i.e., endogenous) promoters canbe employed to direct expression of the nucleic acids of the presentinvention. These promoters can also be used, for example, in recombinantexpression cassettes to drive expression of antisense nucleic acids toreduce, increase, or alter concentration and/or composition of theproteins of the present invention in a desired tissue. Thus, in someembodiments, the nucleic acid construct will comprise a promoterfunctional in a plant cell, such as in Zea mays, operably linked to apolynucleotide of the present invention. Promoters useful in theseembodiments include the endogenous promoters driving expression of apolypeptide of the present invention.

In some embodiments, isolated nucleic acids which serve as promoter orenhancer elements can be introduced in the appropriate position(generally upstream) of a non-heterologous form of a polynucleotide ofthe present invention so as to up or down regulate expression of apolynucleotide of the present invention. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution (see, Kmiec, U.S. Pat. No. 5,565,350 and Zarling et al.,U.S. Pat. No. 5,763,240), or isolated promoters can be introduced into aplant cell in the proper orientation and distance from a gene of thepresent invention so as to control the expression of the gene. Geneexpression can be modulated under conditions suitable for plant growthso as to alter the total concentration and/or alter the composition ofthe polypeptides of the present invention in plant cell. Thus, thepresent invention provides compositions, and methods for making,heterologous promoters and/or enhancers operably linked to a native,endogenous (i.e., non-heterologous) form of a polynucleotide of thepresent invention.

In accordance with other exemplary embodiments, the expression cassettesof the present invention may further include an enhancer element, apolyadenylation region, an intron enhancement element, a selectablemarker, and/or a terminator element.

The expression cassettes of the invention can be used to confer abioticstress-tolerance on essentially any plant. In particular, the inventionhas use in monocots, such as cereal plants, for example, from the generaAvena, Hordeum, Oryza, Secale, Sorghum, Triticum, and Zea. The inventionalso has use over a broad range of plants, including species from thegenera Asparagus, Atropa, Brassica, Citrus, Citrullus, Capsicum,Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus,Heterocallis, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus,Manihot, Majorana, Medicago, Nicotiana, Panieum, Pannesetum, Persea,Pisum, Pyrus, Prunus, Raphanus, Senecio, Sinapis, Solanum, Trigonella,Vitis, and Vigna. In some embodiments of the invention, the expressioncassettes of the invention are used to confer drought-tolerance. In someembodiments of the invention, the expression cassettes of the inventionare used to confer salt-tolerance.

V. Plant Transformation

Once an expression cassette comprising a polynucleotide of the presentinvention has been constructed, any technique now known or hereafterdevised by those skilled in the art may be used to introduce thepolynucleotide into a plant. See, for example, protocols described inAmmirato et al., Handbook of Plant Cell Culture—Crop Species. MacmillanPubl. Co., 1984. Shimamoto et al., Nature 338:274-276, 1989; Fromm etal., Bio/Technology 8:833-839, 1990; and Vasil et al., Bio/Technology8:429-434, 1990.

Transformation and regeneration of plants is generally known in the art,and the selection of the most appropriate transformation technique for aparticular embodiment of the invention may be determined by thepractitioner. Suitable methods may include, but are not limited to:electroporation of plant protoplasts; liposome-mediated transformation;polyethylene glycol (PEG) mediated transformation; transformation usingviruses; micro-injection of plant cells; micro-projectile bombardment ofplant cells; vacuum infiltration; and Agrobacterium tumeficiens mediatedtransformation. Transformation means introducing a nucleotide sequencein a plant in a manner to cause stable or transient expression of thesequence. Examples of these methods in various plants include: U.S. Pat.Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615;5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and5,610,042.

Following transformation, plants preferably are selected using adominant selectable marker incorporated into the transformation vector.Typically, such a marker will confer antibiotic or herbicide resistanceon the transformed plants, and selection of transformants can beaccomplished by exposing the plants to appropriate concentrations of theantibiotic or herbicide.

The cells, which have been transformed, may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.,Plant Cell Reports 5:81-84, 1986. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having the desired phenotypic characteristicidentified. Two or more generations may be grown to ensure that thesubject phenotypic characteristic is stably maintained and inherited andthen seeds harvested to ensure the desired phenotype or other propertyhas been achieved.

VI. Production of Transgenic Plants by Genetic Crosses

The present invention relates to methods of generating abioticstress-tolerant plants by transferring a nucleic acid of the presentinvention, from a donor plant into a recipient plant strain which is notabiotic stress-tolerant, thus conferring the trait of abioticstress-tolerance to the recipient strain.

Accordingly, one method to accomplish such a transfer is byintrogression of a nucleic acid sequence conferring or contributing tothis trait from an abiotic stress-tolerant donor plant into a recipientplant that is not abiotic stress-tolerant by crossing said plants. Thistransfer may thus suitably be accomplished by using traditional breedingtechniques.

In one method, a donor plant that exhibits abiotic stress-tolerance andcomprising a nucleic acid of the present invention, is crossed with aplant that is not abiotic stress-tolerant and preferably exhibitscommercially desirable characteristics, such as, heavier grains, alonger grain-filling period, and sturdier stems, etc. The resultingplant population (representing the F1 hybrids) is then self-pollinatedand seeds are obtained (F2 seeds). The F2 seeds can then be screened forabiotic stress-tolerance as by any of the methods described herein.

Inbred abiotic stress-tolerant plant lines can be developed using thetechniques of recurrent selection and backcrossing, selfing and/ordihaploids or any other technique used to make parental lines. In amethod of selection and backcrossing, abiotic stress-tolerance trait canbe introgressed into a target recipient plant (which is called therecurrent parent) by crossing the recurrent parent with a first donorplant (which is different from the recurrent parent and referred toherein as the “non-recurrent parent”). The recurrent parent is a plantthat is not abiotic stress-tolerant and possesses commercially desirablecharacteristics.

The non-recurrent parent exhibits abiotic stress-tolerance andcomprising a nucleic acid of the present invention, wherein theexpression of the polypeptides of the present invention is enhanced ascompared to a plant that is not abiotic stress-tolerant. Thenon-recurrent parent can be any plant variety or inbred line that iscross-fertile with the recurrent parent. The progeny resulting from across between the recurrent parent and non-recurrent parent arebackcrossed to the recurrent parent. The resulting plant population isthen screened. F1 hybrid plants that comprise the requisite nucleic acidof the present invention are then selected and selfed and selected for anumber of generations in order to allow for the plant to becomeincreasingly inbred. This process of continued selfing and selection canbe performed for two to five or more generations. The result of suchbreeding and selection is the production of lines that are geneticallyhomogenous for the genes associated with abiotic stress-tolerance aswell as other genes associated with traits of commercial interest.

Abiotic stress-tolerance can be assayed according to any of a number ofwell-know techniques. The determination that a plant modified accordingto a method of the invention has increased tolerance to astress-inducing condition can be made by comparing the treated plantwith a control (reference) plant using well-known methods. For example,a plant having increased tolerance to salt stress can be identified bygrowing the plant on a medium such as soil that contains salt at a levelmore than about 100% of the amount of salt in the medium on which thecorresponding reference plant is capable of growing. Advantageously, aplant treated according to a method of the invention can grow on amedium or soil containing salt at a level of at least about 110%,preferably at least about 150%, more preferably at least about 200%, andoptimally at least about 400% of the level of salt in the medium or soilon which a corresponding reference plant can grow. In particular, such atreated plant can grow on medium or soil containing at least 40 mM,generally at least 100 mM, particularly at least 200 mM, and preferablyat least 300 mM salt, including, for example, a water soluble inorganicsalt such as sodium sulfate, magnesium sulfate, calcium sulfate, sodiumchloride, magnesium chloride, calcium chloride, potassium chloride, orthe like; salts of agricultural fertilizers, and salts associated withalkaline or acid soil conditions; particularly NaCl.

Drought-tolerance can be determined by any of a number of standardmeasures including turgor pressure, growth, yield and the like. Forexample, a plant having increased tolerance to drought can be identifiedby growing the plant under conditions in which less than the optimalamount of water is provided to the plant through precipitation and/orirrigation. Particularly, a plant having increased tolerance to droughtcan be identified by growing the plant on a medium such as soil thatcontains less water than the medium on which the corresponding referenceplant is capable of growing. Advantageously, a plant treated accordingto a method of the invention can grow on a medium or soil containingsalt at a level of less than about 90%, preferably less than about 80%,more preferably less than about 50%, and optimally less than about 20%of the amount of water in the medium or soil on which a correspondingreference plant can grow. Alternatively, a plant having increasedtolerance to drought can be identified by its ability to recover fromdrought when rehydration is provided after a period of drought.Advantageously, a plant treated according to a method of the inventioncan recover when rehydration is provided after a period of at least 3days drought, at least 5 days drought, preferably at least 7 daysdrought, more preferably at least about 10 days drought, and optimallyat least about 18 days drought.

Water use efficiency can be determined by evaluating the amount of drybiomass that a plant accumulates (which can be vegetative, reproductive,or both, depending on the yield component(s) of interest) per unit wateravailable to the plant. A plant having enhanced water use efficiencywill have a greater amount of dry biomass accumulation per unit wateravailable than the corresponding reference plant grown under the sameconditions. Water use efficiency at the leaf or plant scale refers tothe ratio between the net CO2 assimilation rate and the transpirationrate, usually measured over a period of seconds or minutes. A plant withenhanced water use efficiency will have higher yields (such as 1-5%,5-10%, 10-15% higher) under restricted water conditions compared to thecorresponding reference plant grown under the same conditions.

Heat tolerance can be determined by evaluating the amount of dry biomassthat a plant accumulates (which can be vegetative, reproductive, orboth, depending on the yield component(s) of interest) relative toincreasing temperatures. A plant having enhanced heat tolerance willhave higher yields (such as 1-5%, 5-10%, 10-15% higher) under increasedtemperature conditions (such as 1° C., 2° C., 3° C., 4° C., etc.)compared to the corresponding reference plant grown under the sameconditions.

Once the appropriate selections are made, the process is repeated. Theprocess of backcrossing to the recurrent parent and selecting forabiotic stress-tolerance is repeated for approximately five or moregenerations. The progeny resulting from this process are heterozygousfor one or more genes that encode for abiotic stress-tolerance. The lastbackcross generation is then selfed in order to provide for homozygouspure breeding progeny for abiotic stress-tolerance.

The abiotic stress-tolerant inbred plant lines described herein can beused in additional crossings to create further abiotic stress-toleranthybrid plants. For example, a first abiotic stress-tolerant inbred plantof the invention can be crossed with a second inbred plant possessingother commercially desirable traits. The second inbred plant line may ormay not also display abiotic stress-tolerance.

VII. Examples Example 1 Cloning and Sequence Analysis of Rice HPLs

Genomic DNA isolated from rice L. cv Nipponbare was used for PCR-basedamplification of these genes using the following gene-specificoligonucleotides: OsHPL1 (Forward:5′-ATAGATATCGCATGCATGGCGCCGCCGCGAGCCAACTCCG-3′ and Reverse:5′-ATATACGTACTGCAGCGCGCGCCGCCGCTTGACACTATTA-3′), OsHPL2 (Forward:5′-ATAGATATCGCATGCATGGCGCCACCGCCAGTGAACTCCG-3′ and Reverse:5′ATATACGTACTGCAGGCACGTGACGTCGACGTGCGTGCTA-3′), and OsHPL3 (Forward:5′-ATAGATATCGCATGCATGGTGCCGTCGTTCCCGCAGCCGG-3′ and Reverse:5′-ATATACGTACTGCAGGAGAGAATGGCGGCAGCAAAGCTTA-3′). For each amplification,30 PCR cycles were carried out using a Gene Amp PCR system 9700 (AppliedBiosystems) in a 25 μL reaction mix containing 10 mM Tris-HCl (pH 8.3),50 mM KCl, 1.5 mm MgCl₂, 4% dimethyl sulfoxide (DMSO), 100 μm of eachdNTP, 500 nM of each forward and reverse primer, 0.625 units of Taq DNApolymerase (Invitrogen), and 50 ng of the genomic DNA. Amplification wasconducted at 94° C. for 1 min, 94° C. for 30 s, 55° C. for OsHPL1, 63°C. for OsHPL2, and 55° C. for OsHPL3 for 1 mM, 72° C. for 90 s, and a10-min extension step at 72° C. The amplified products were resolved byelectrophoresis on a 1% (w/v) agarose gel. The band corresponding toeach full-length gene was cut, purified using QIAquick Gel extractionkit (Qiagen), and cloned in pCR 2.1-TOPO Vector (Invitrogen) accordingto the manufacturer's instructions. The identities of these clones wereconfirmed by DNA sequencing. All DNA as well as polypeptide sequenceanalyses were performed using Vector NTI advance program 9 (Invitrogen).

Example 2 Arabidopsis Transformation of Three Rice HPLs

Green fluorescent protein (GFP) fusions for stable expression wereconstructed by cloning the PCR-amplified, TOPO-cloned, andEcoRI-/BamHI-digested fragments of the full length of all three riceHPLs into the EcoRI/BamHI site of pEZS-NLGFP. Primers were designed toeliminate stop codons and fuse the coding sequences to the 5′ end of theGFP gene. For OsHPL1, the primers used were: Forward:5′-ATA-GAATTCATGGCGCCGCCGCGAG-3′ and Reverse:5′-ATAGGATCCGCTA-CTCCGCGCCGCGCG-3′. For OsHPL2, the primers used were:Forward: ATAGAATTCATGGCGCCACCGCCAGT-3′ and Reverse:5′-ATAGGATCC-GCTCCCGACGACGCCCGT-3′. OsHPL3 was amplified using thefollowing primers: Forward: 5′-ATAGAATTCATGGTGCCGTCGTTCCC-3′ andReverse: 5′-ATAGGATCCGCGCTGGGAGTGAGCTCCC-3′. To generate OsHPL3-TP (HPL3minus the first 15 amino acids of the plastid transit peptide at theamino terminus of the protein), OsHPL3 cDNA was amplified (Forward:5′-CCGGCCAATACCGGGG-3′ and Reverse 5′-TTAGCTGGGAGTGAGCTC-3′). PCRamplifications were conducted as described above with a T_(m)=55° C.used for all genes amplified. GFP fusions for Arabidopsis transformationwere created by subcloning the OsHPL1, OsHPL2, and OsHPL3 open readingframes from pEZS-NLGFP into a binary vector using NotI restriction siteswith the GFP gene at the C terminus of each gene. For OsHPL3-TP, the PCRproduct was cloned into Gateway pENTER vector, according to themanufacturer's recommendation. The construct was then fully sequenced,and the pB7WGF2-OsHPL3-15AA TP construct was generated in arecombination reaction between the entry clone pENTR OsHPL3-15AA TP andpB7WGF2 vector. The constructs were verified by sequencing, introducedinto Agrobacterium EHA101 strain, and used to transform Arabidopsisplants by using the floral-dip method (Clough, S. J. and Bent, A. F.,Plant J. 16: 735-743, 1998). The T1 plants were germinated on soil.Selection of transgenics was by treating 10- to 12-d-old seedlings with1:1,000 Finale (the commercial product that is 5.78% glufosinateammonium) twice a week. The localization of OsHPL1, HPL2, and HPL3-TPoutside the plastid and OsHPL3 inside the plastid was confirmed intransformed plants.

Example 3 Expression of Rice HPL1 and/or HPL2 in Arabidopsis ConfersSalt-Tolerance

Enhanced tolerance of both HPL1 (p≦0.003) and HPL2 (p≦0.001) lines tosalt-stress was observed, as measured by the survival rate of plantsexposed to 200 mM NaCl for five days (FIG. 1). Col-0, a natural hpl nullmutant, was used as a control. Homozygous lines expressing the correctedversion of the HPL genes under endogenous promoter of Col-0 were used.

In a second experiment, five week old HPL2 and Col-0 plants weresubjected to salt treatment for three weeks followed by recovery for tendays. Plants were watered one every three days with a nutrient solution(modified Spalding solution). The volume of liquid added was such thatit allowed for 1/3 leaching volume. For the pot sizes used, 50-75 ml ofnutrient solution was added per pot. When plants were treated with salt,they were watered with the nutrient solution plus 100 mM NaCl once everythree days. During the recovery period, plants were watered with thenutrient solution every three days. The HPL2 line had a greater survivalrate following 100 mM salt stress than the Col-0 line when plants weregrown on either Sunshine Mix #3 (67% versus 50%) or a 50/50 mix ofSunshine Mix #3 and Profile Green (31% versus 21%).

Example 4 Expression of Rice HPLs Outside the Plastid in ArabidopsisConfers Drought-Tolerance

Enhanced tolerance of HPL1, HPL2 and HPL3-TP (HPL3 minus the 15 aminoacids of the plastid transit peptide with localization of the enzymeoutside the plastid) lines to drought-stress was observed (p≦0.001,0.004, and 0.001, respectively), as measured by the survival rate ofplants after ten days of water withdrawal (FIG. 2). Col-0, a natural hplnull mutant, was used as a control. In contrast to the lines in whichHPL was localized outside the plastid, survival of the HPL3 line (enzymewas localized in the plastid) did not differ from the Col-0 line. Plantswere grown in individual pots containing the same amount of soil. Allpots were watered with the same amount of water. When plants were 2.5weeks old (all plants had 8-10 true leaves), water was withheld for 9days, a time point at which about 50% of Col-0 plants looked dead.Subsequently plants were watered excessively and were left to recoverfor 5 days before further analysis. Three independent experiments werecarried out. In each experiment, each line was represented by 10-14plants.

Example 5 Micro-array Analysis of Arabidopsis Lines Expressing Rice HPL1and HPL3

Gene expression levels was evaluated in leaves from Arabidopsis linesexpressing rice HPL1 and rice HPL3 compared to Col-0. RNA was extractedfrom leaves of three-week old plants grown in a growth chamber understandard conditions (16-hours light/8-hours dark cycle at 22° C.). Thethree biological samples (HPL1, HPL3, Col-0) were run in duplicate usingArabidopsis chips from Agilent Technologies. While several differenceswere observed in gene expression levels between the HPL1 line and theHPL3 line compared to Col-0, of particular interest was the observationthat several sequences associated with heat shock proteins and heatshock transcription factors were increased to a greater degree in theHPL1 line than in the HPL3 line (see TABLE 1).

TABLE 1 Examples of differentially expressed heat shock associatedproteins in the HPL1 line and the HPL3 line compared to Col-0. Fold FoldChange Change Brief Sequence Description HPL1 HPL3 At2g26150: heat shocktranscription factor family 8.5 2.22 protein At5g51440: small heat shockprotein (HSP23.5-M) 8.37 2.15 At3g12580: heat shock protein 70 5.65 1.8 At1g07400: 17.8 kDa class I heat shock protein 5.23 1.68 At2g20560: DNAJheat shock family protein 2.82 1.48 SP|Q9UDY4 At1g74310: heat shockprotein 101 (HSP101) 2.74 ND At5g37670: 15.7 kDa class I-related smallheat shock 2.44 1.41 protein-like (HSP15.7-CI) At4g11660: heat shocktranscription factor 7 (HSTF7) 2.24 1.33 At3g14200: DNAJ heat shockN-terminal domain- 2 ND containing protein At1g56410: heat shock cognate70 kDa protein 1.95 ND

One of these sequences, heat shock protein 101, was upregulated2.74-fold in HPL1 compared to Col-0 but unchanged (ND) compared to Col-0in the HPL3 line. Heat shock protein 101 has been shown recently to playan important role in conferring tolerance to heat (Tonsor et al., Mol.Ecol. 17(6):1614-1626, 2008). These data provide evidence thatoverexpression of HPL genes outside the plastid may provide protectionagainst damage due to increasing temperature leading to enhanced heattolerance.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Informal Sequence Listing

(HPL1, AK105964) SEQ ID NO: 1 1gtggctgtga cgatccgaca cctgcacgct agtacgtagt gcgtatacgt agccagtacc 61ctactcccgt ccatggcgcc gccgcgagcc aactccggcg acggtaacga cggcgccgtc 121ggagggcaga gcaagctctc gccgtcgggc ctgctgatac gcgagattcc gggcggctac 181ggcgtgccct tcctctcgcc gctgcgcgac cgcctcgact actattactt ccagggcgcc 241gacgagttct tccgctcacg cgtcgcccgc cacggcggcg ccaccgtgct ccgcgtcaac 301atgccgcccg gccccttcct cgccggcgac ccccgcgtcg tcgccctcct cgacgcgcgc 361agcttccgcg tcctcctcga cgactccatg gtggacaagg ccgacacgct cgacggcacc 421ttcatgccgt cgctcgcgct cttcggcggc caccgcccgc tcgccttcct cgacgccgcc 481gaccctcgcc acgccaagat caagcgcgtc gtgatgtcgc tcgccgcggc gaggatgcac 541cacgtcgcgc cggcgttccg cgccgccttc gccgccatgt tcgacgaggt cgacgccggc 601ctcgtcgccg gcggccccgt cgagttcaac aagctcaaca tgcggtacat gctcgacttc 661acctgcgccg cgctgttcgg cggcgcgccg ccgagcaagg ccatgggcga cgctgccgtg 721acgaaggcgg tgaagtggct catcttccag cttcacccgc tcgccagcaa ggtcgtcaag 781ccgtggccgc tggaggacct cctcctccac accttccgcc tgccgccgtt cctggtgcgc 841cgcgagtacg gcgagatcac ggcgtacttc gccgccgccg ccgcggccat cctcgacgac 901gccgagaaga accacccggg aatcccgcgc gacgagctcc tccacaacct cgtgttcgtc 961gccgtcttca acgcctacgg cggcttcaag atcttcctgc cacacatcgt caagtggctc 1021gcccgcgccg gcccggagct ccacgccaag ctagcctccg aggtccgcgc cgccgcgccc 1081gccggcggcg gcgagatcac catctccgcc gtggagaagg agatgccgct ggtgaagtcg 1141gtggtgtggg aggcgctgcg catgaacccg ccggtggagt tccagtacgg gcgcgcgcgg 1201cgcgacatgg tcgtcgagag ccacgacgcg gcgtacgagg tccgcaaggg ggagctgctg 1261ttcgggtacc agccgctcgc cacccgcgac gagaaggtgt tcgaccgcgc cggcgagttc 1321gtccccgacc ggttcgtctc cggcgccgga agcgccgccc ggccgctgct ggagcacgtg 1381gtgtggtcga acgggccgga gaccgggacg ccatcggagg ggaacaagca gtgccccggg 1441aaggacatgg tggtggcggt ggggcggctg atggtggcgg ggctgttccg gcggtacgac 1501acgttcgccg ccgacgtgga ggagctgccg cttgagccgg tggtcacgtt cacgtcgctg 1561acccgcgccg ccgacggcga cggcgccgcg cggcgcggag tataatagtg tcaagcggcg 1621gcgcgcgtga gcggcgagtg ttggtgcggc gacgacgctg tccatgcatg gtcgctgtca 1681gttggtcaga tttgcatgga tttctttttt ctttgaccta aaaaaattgg gaaaaaggtg 1741tactttcgcg tgcttgtggg ggcaggttct taagtatagg gattcggttt gtcattgtgt 1801gaagttcaat acgatgtttg aagttgaata aaattatgtg cgttcctcgt ggttttSEQ ID NO: 2MAPPRANSGDGNDGAVGGQSKLSPSGLLIREIPGGYGVPFLSPLRDRLDYYYFQGADEFFRSRVARHGGATVLRVNMPPGPFLAGDPRVVALLDARSFRVLLDDSMVDKADTLDGTFMPSLALFGGHRPLAFLDAADPRHAKIKRVVMSLAAARMHHVAPAFRAAFAAMFDEVDAGLVAGGPVEFNKLNMRYMLDFTCAALFGGAPPSKAMGDAAVTKAVKWLIFQLHPLASKVVKPWPLEDLLLHTFRLPPFLVRREYGEITAYFAAAAAAILDDAEKNHPGIPRDELLHNLVFVAVFNAYGGFKIFLPHIVKWLARAGPELHAKLASEVRAAAPAGGGEITISAVEKEMPLVKSVVWEALRMNPPVEFQYGRARRDMVVESHDAAYEVRKGELLFGYQPLATRDEKVFDRAGEFVPDRFVSGAGSAARPLLEHVVWSNGPETGTPSEGNKQCPGKDMVVAVGRLMVAGLFRRYDTFAADVEELPLEPVVTFTSLTRAADGDGAARRGV (HPL2, AK107161)SEQ ID NO: 3 1ctcctcgaac caacccaaca caacacttgc acttgcacta cgtactctca tttcatccgc 61tcccggccgg caatggcgcc accgccagtg aactccggcg acgccgccgc cgccgccacg 121ggagagaaga gcaagctctc gccgtcgggc ctccccatac gcgagatacc cggcggctac 181ggcgtgccct tcttctcgcc gctgcgcgac cgcctcgact acttctactt ccagggcgcc 241gaggagtact tccgatcacg cgtcgcccgc cacggcggcg ccaccgtgct ccgcgtcaac 301atgccgcccg gccccttcat ctccggcaac ccccgcgtcg tcgccctcct cgacgcgcgc 361agcttccgcg tcctcctcga cgactccatg gtggacaagg ccgacacgct cgacggcacc 421tacatgccgt cgcgcgcgct cttcggcggc caccgcccgc tcgccttcct cgacgccgcc 481gacccgcgcc acgccaagat caagcgcgtc gtgatgtcgc tcgccgccgc gcggatgcac 541cacgtcgcgc cggcgttccg cgccgccttt gccgccatgt tcgacgccgt cgaggccggc 601ctcggcgccg ccgtcgagtt caacaagctc aacatgaggt acatgctcga cttcacctgc 661gccgcgctgt tcggcggcga gccgccgagc aaggtggtcg gcgacggcgc cgtgacgaag 721gccatggcgt ggctcgcgtt ccagctgcac ccgatcgcga gcaaggtcgt caagccatgg 781ccgctcgagg agctactcct gcacaccttc tccctgccgc cgttcctggt gcggcgtggc 841tacgccgacc tgaaggcgta cttcgccgac gccgccgcgg ccgtcctcga cgacgccgag 901aagagccaca cgggaatccc gcgcgacgag ctcctcgaca accttgtgtt cgtcgccatt 961ttcaacgcct tcggcggctt caagatcttc ctgccacaca tcgtcaagtg gctcgcccgc 1021gccggcccgg agctccacgc caagcttgcc accgaggtcc gcgccaccgt gcccaccggc 1081gaggacgacg gcatcaccct cgccgccgtc gagcggatgc cgctggtgaa gtcggtggtg 1141tgggaggcgc tgcgcatgaa cccgccggtg gagttccagt acggccacgc gcggcgcgac 1201atggtggtcg agagccacga cgcggcgtac gaggtgcgca agggggagat gctgttcggc 1261taccagccgc tcgccacccg cgacgagaag gtgttcgacc gcgccggcga gttcgtcgcc 1321gaccggttcg tcgccggcgg cgccgccggc gaccggccgc tgctggagca cgtggtgtgg 1381tcgaacgggc cggagacgag ggcgccatcg gaggggaaca agcagtgccc cgggaaggac 1441atggtggtgg cggtggggcg gctgatggtg gcggagctgt tccggcggta cgacacgttc 1501gccgccgacg tggtggaggc gccggtggag ccggtggtga cgttcacgtc gctgacacgg 1561gcgtcgtcgg gatagcacgc acgtcgacgt cacgtgcgcg ccgtgctgtg atttagtact 1621gtactaggtt ggtggatgtt ttaattgcgt ggttaattat taatcacgca taaagtatta 1681atcatgtttt atcatctaac aacaatgaaa atattaatca t SEQ ID NO: 4MAPPPVNSGDAAAAATGEKSKLSPSGLPIREIPGGYGVPFFSPLRDRLDYFYFQGAEEYFRSRVARHGGATVLRVNMPPGPFISGNPRVVALLDARSFRVLLDDSMVDKADTLDGTYMPSRALFGGHRPLAFLDAADPRHAKIKRVVMSLAAARMHHVAPAFRAAFAAMFDAVEAGLGAAVEFNKLNMRYMLDFTCAALFGGEPPSKVVGDGAVTKAMAWLAFQLHPIASKVVKPWPLEELLLHTFSLPPFLVRRGYADLKAYFADAAAAVLDDAEKSHTGIPRDELLDNLVFVAIFNAFGGFKIFLPHIVKWLARAGPELHAKLATEVRATVPTGEDDGITLAAVERMPLVKSVVWEALRMNPPVEFQYGHARRDMVVESHDAAYEVRKGEMLFGYQPLATRDEKVFDRAGEFVADRFVAGGAAGDRPLLEHVVWSNGPETRAPSEGNKQCPGKDMVVAVGRLMVAELFRRYDTFAADVVEAPVEPVVTFTSLTRASSG (HPL3, AY340220)SEQ ID NO: 5 1tagagtcagt gtcataacgc aagctaccac acgtagctga taagtccgat cgtcgccgcg 61cgccgcgcca tggtgccgtc gttcccgcag ccggccagtg cggcggcggc gacgcggcca 121ataccgggga gctacggccc gccgctgctc ggcccgctcc gcgaccgcct cgactacttc 181tggttccagg gccccgacga cttcttccgc cgccgcgccg ccgaccacaa gagcaccgtg 241ttccgcgcca acatcccgcc caccttcccc ttcttcctcg gcgtcgaccc gcgcgtcgtc 301gccgtcgttg atgccgccgc cttcaccgcg ctcttcgacc cggccctcgt cgacaagcgc 361gacgtcctca tcggccccta cgtccccagc ctcgccttca cccgcggcac ccgcgtcggc 421gtctacctcg acacccagga ccccgaccac gcccgcacca aggccttctc catcgacctc 481ctccgccgcg ccgcccgcaa ctgggccgcc gagctccgcg ccgccgtcga cgacatgctc 541gccgccgtcg aggaagacct caacagggcc cctgaccccg ccgccgcctc cgccagctac 601ctcatcccgc tccagaagtg catcttccgc ttcctctgca aggcgctcgt cggcgccgac 661ccggcggcgg acggcctcgt cgaccgcttc ggcgtgtaca tcctcgacgt gtggctggcg 721ttgcagctgg tgccgacgca gaaggtgggc gtcatcccgc agccgctgga ggagctcctg 781ctccactcct tcccgctgcc gtcgttcgtc gtcaagcccg ggtacgacct cctctaccgc 841ttcgtggaga agcacggcgc cgccgccgtg tccatcgctg agaaggagca cggcatcagc 901aaggaggagg ccatcaacaa catcctcttc gtgctcggct tcaacgcgtt cggcggcttc 961tcggtgttcc tgccgttcct ggtcatggag gtcggcaagc ccggccggga agacctgcgg 1021cggcggctgc gggaggaggt gcgccgcgtg ctgggcggcg gcgacggcgg cgaggccggg 1081ttcgcggcgg tgagggagat ggcgctggtg cggtcgacgg tgtacgaggt gctccggatg 1141cagccgccgg tgccgctgca gttcgggcgg gcgcggcgag acttcgtgct gcggtcgcac 1201ggcggcgcgg cgtacgaggt gggcaagggc gagctgctgt gcgggtacca gccgctggcc 1261atgcgcgacc cggcggtgtt cgaccggccg gaggagttcg cgccggagag gttcctcggc 1321gacgacggcg aggcgctgct gcagtacgtg tactggtcca acgggccgga gaccggcgag 1381ccgtcgccgg ggaacaaaca gtgtgccgcc aaggaggtgg tcgtcgccac cgcgtgcatg 1441ctcgtcgccg agcttttccg gcggtacgac gacttcgaat gcgacggcac ctccttcacc 1501aagctcgaca agcgggagct cactcccagc taagctttgc tgccgccatt ctctcactcg 1561atctccatgc acatatgcat gaagaaatta attaaattca agttgctagc tccatttttt 1621ctctttgagc tgctgataaa aaaacatctc tattcttctg tgcaataagc caataattaa 1681gcattaatca gagcgtacaa gtaaaaattg ttttcactgt tttatgtgga tunderlined sequences are deleted to prevent transport of encoded polypeptideinto the plastid SEQ ID NO: 6 MVPSFPQPASAAAATRPIPGSYGPPLLGPLRDRLDYFWFQGPDDFFRRRAADHKSTVFRANIPPTFPFFLGVDPRVVAVVDAAAFTALFDPALVDKRDVLIGPYVPSLAFTRGTRVGVYLDTQDPDHARTKAFSIDLLRRAARNWAAELRAAVDDMLAAVEEDLNRAPDPAAASASYLIPLQKCIFRFLCKALVGADPAADGLVDRFGVYILDVWLALQLVPTQKVGVIPQPLEELLLHSFPLPSFVVKPGYDLLYRFVEKHGAAAVSIAEKEHGISKEEAINNILFVLGFNAFGGFSVFLPFLVMEVGKPGREDLRRRLREEVRRVLGGGDGGEAGFAAVREMALVRSTVYEVLRMQPPVPLQFGRARRDFVLRSHGGAAYEVGKGELLCGYQPLAMRDPAVFDRPEEFAPERFLGDDGEALLQYVYWSNGPETGEPSPGNKQCAAKEVVVATACMLVAELFRRYDDFECDGTSFTKLDKRELTPSunderlined sequences are deleted to prevent transport of polypeptide intothe plastid

1. A method of preparing a plant tolerant to abiotic stress, the method comprising: (a) introducing into a population of plants a recombinant expression cassette comprising a hydroperoxide lyase (HPL) polynucleotide encoding a HPL enzyme; and (b) selecting a plant that is tolerant to abiotic stress; wherein the HPL enzyme comprises (L/I)-(F/C)-G-(Y/F)-(Q/R)-(P/K) and (N/D)-K-(Q/D-C-(A/P)-(G/A)-K-(D/N).
 2. The method of claim 1, wherein the HPL enzyme is localized outside the plastid when expressed in the population of plants.
 3. The method of claim 1, wherein the HPL enzyme recognizes 9-hydroperoxy-octadecatrienoic acid (9-HPOT) or 9-hydroperoxy-octadecadienoic acid (9-HPOD).
 4. The method of claim 1, wherein the HPL enzyme recognizes 13-hydroperoxy-octadecatrienoic acid (13-HPOT) or 13-hydroperoxy-octadecadienoic acid (13-HPOD).
 5. The method of claim 1, wherein the HPL enzyme is localized outside the plastid when expressed in the population of plants, and wherein the HPL enzyme recognizes 9-hydroperoxy-octadecatrienoic acid (9-HPOT) or 9-hydroperoxy-octadecadienoic acid (9-HPOD).
 6. The method of claim 5, wherein the HPL enzyme further recognizes 13-hydroperoxy-octadecatrienoic acid (13-HPOT) or 13-hydroperoxy-octadecadienoic acid (13-HPOD).
 7. The method of claim 1, wherein the HPL enzyme has an amino acid sequence at least 90% identical to SEQ ID NO. 2, 4 or
 6. 8. A method of preparing a plant tolerant to abiotic stress, the method comprising: (a) introducing into a population of plants a recombinant expression cassette comprising a hydroperoxide lyase (HPL) polynucleotide encoding a HPL enzyme wherein the HPL enzyme has an amino acid sequence at least 90% identical to SEQ ID NO. 2, 4 or 6; and (b) selecting a plant that is tolerant to abiotic stress, wherein the HPL enzyme is localized outside the plastid when expressed in the population of plants.
 9. The method of claim 1, wherein the step of introducing is carried out by a sexual cross.
 10. The method of claim 1, wherein the step of introducing is carried out using micro-projectile bombardment.
 11. The method of claim 1, wherein the HPL polynucleotide is SEQ ID NO. 1, 3 or
 5. 12. The method of claim 1, wherein the plant is Oryza sativa.
 13. The method of claim 1, wherein the abiotic stress is drought.
 14. The method of claim 1, wherein the abiotic stress is salt.
 15. The method of claim 1, wherein the HPL polynucleotide is operably linked to a promoter.
 16. The method of claim 15, wherein the promoter is constitutive or inducible.
 17. A transgenic plant comprising a recombinant expression cassette, wherein the recombinant expression cassette comprises a HPL polynucleotide encoding a HPL enzyme having an amino acid sequence at least 90% identical to SEQ ID NO. 2, 4 or 6, with a proviso that the plant is not Arabidopsis.
 18. The transgenic plant of claim 17, wherein the plant is Oryza sativa.
 19. A transgenic seed from the transgenic plant of claim
 18. 20. The transgenic seed of claim 19, wherein the seed is a transgenic Oryza sativa seed. 